Portable Detection of Melamine in Milk Using a Personal Glucose Meter Based on an in Vitro Selected Structure-Switching Aptamer

Abstract

Melamine detection in milk and other foods has attracted much attention since the discovery that melamine-adulterated food causes severe kidney damage. Although many methods have been developed to detect melamine, few methods can provide quantitative results using an affordable and portable device that is suitable for home use or field application. To achieve this goal, we herein report the first in vitro selection of a melamine responsive aptamer using a structure-switching method. A personal glucose meter (PGM) based melamine sensor was designed and subsequently tested using the newly isolated aptamer. Conversion of melamine concentration to glucose amount was achieved by including an invertase-conjugated DNA that is complementary to part of the aptamer. Melamine binding triggers the release of the invertase-DNA conjugate, which hydrolyzes sucrose into glucose. The glucose produced is then measured directly using an off-the-shelf PGM. The described sensor shows high selectivity for melamine against several closely related melamine analogues, such as cyanuric acid, ammeline, and ammelide, and has low detection limits of 0.33 μM (or 41.1 ppb) in buffer and 0.53 μM (or 67.5 ppb) in 80% whole milk without any pretreatment. The detection limits meet the threshold of 2.5 ppm for non-infant-formula products and 1 ppm for melamine in infant milk products as defined by the U.S. Food and Drug Administration (FDA). In addition to the PGM sensor demonstrated here, the same aptamer can be converted into other types of sensors with different signal outputs, allowing portable detection of melamine under a variety of conditions.

Point-of-care diagnostics for niche applications

“…The cost of the technologies included in portable tests is going down fast. Tests that use glucose meters (Gu et al, 2015) or cell phones (Yu et al, 2015; Ludwig et al, 2015) to analyze milk samples are in the research phase. Fabrication technologies, including cloned reagents, especially synthetic antibodies, ink jet printing of biologics and optoelectronic circuits, organic optics, and roll-to-roll fabrication and assembly of devices will bring the cost down and enable the production of more robust and disposable tests.…”

Transforming the blood glucose meter into a general healthcare meter for in vitro diagnostics in mobile health

“…Compared to antibodies, aptamers and DNAzymes have distinct advantages originating from their nucleic acid properties: 1) they can be synthesized in the test tubes at a much lower cost than that of antibodies; 2) they can be readily conjugated to many signal transducing elements, such as fluorophores, nanomaterials and enzymes at an exact location; 3) they are more stable than antibodies at room temperature and during storage; 4) unlike antibodies, they can be denatured during manufacturing process and storage and can regain binding or catalytic activity after renaturation at physiological condition before sensing applications; 5) since the DNA and RNA can be synthesized chemically, they have minimal batch-to-batch variations, and 6) they have been shown to bind small molecules such as metal ions much more selectively than those of antibodies. These properties make them excellent molecular recognition elements in developing BGM-based biosensors to quantify a variety of analytical targets, such as cocaine (Xiang and Lu, 2011, Yan et al, 2013, Hou et al, 2014, Zhou et al, 2014b), adenosine (Xiang and Lu, 2011, Liu et al, 2012), ATP (Yan et al, 2013, Hou et al, 2014), dopamine (Hun et al, 2015b), melamine (Gu et al, 2015), myoglobin (Wang et al, 2015a), platelet-derived growth factor-BB (PDGF-BB) (Ma et al, 2014), interferon-gamma (Xiang and Lu, 2011, Lan et al, 2015), uranium (UO 2 2+ ) (Xiang and Lu, 2011, 2013), and lead (Pb 2+ ) (Fu et al, 2013, Xiang and Lu, 2013, Liao and Li, 2014, Zhang et al, 2015a). …”

“…The binding of the target by the aptamer weakens the hybridization and triggers the release of the DNA-conjugated invertase, which can be used for glucose production (Figure 3). For instance, by using the structure-switching SELEX method (Nutiu and Li, 2003, 2004, 2005), a melamine responsive aptamer was selected and then converted to a BGM-based melamine sensor with the assistance of invertase-conjugated DNA (Gu et al, 2015). In addition, the use of purified DNA invertase conjugates from reaction mixture via a biotin-desthiobiotin displacement strategy was demonstrated to further improve the analytical performance of BGM-based sensors (Zhou et al, 2014b).…”

DNA Aptamer-Based Activatable Probes for Photoacoustic Imaging in Living Mice

“…4 Hence, many aptamers have been converted into fluorescent, 5 colorimetric 6 and electronical sensors. 7 Among them, fluorescent sensors remain dominant probes for biomedical applications, 5a−5e,8 because of their high intensity and fast signal response.…”